The objective of the project is to study the determinants of virulence in B. fragilis by: A) defining transferable drug resistance; B) developing a genetic system to obtain mutants lacking suspected virulence factors; and C) developing a phage typing system to investigate the epidemiology of infections caused by B. fragilis. The clindamycin resistance (clin r) transfer factor pBFTM 10 from B. fragilis TM 4003 has been mapped by restriction endonuclease enzymes. A spontaneous clindamycin sensitive derivative of TM 4003 was found to have a plasmid pBFTM delta 1, which lacks a 5.6 Kb area which includes the clin r determinant in part or whole. Heteroduplex analysis of the parent and deleted plasmid reveal that the deleted section contains a small inverted repeat segment. EcoR1 digest fragments of pBFTM 10 have been cloned into E. coli utilizing the vector pMC 1403. A 4.5 Kb fragment which is thought to include the clin r genes is flanked by a 75 pb inverted repeat sequence. This suggests that the clin r genes may be on a transposable element. p32 labelled pBFTM 10 DNA has been used as a probe in the Southern blotting procedure to investigate clin r and tetracycline resistance (tet r) transfer in a plasmid-free strain of B. fragilis TM 230. The hybridization studies support the hypothesis that transfer is due to the presence of a transfer element in donor cell chromosomes which is capable of promoting the transfer of an element by conjugation of it into recipient chromosome. A mutagenic system using nitrosoguanidine has been utilized to isolate the amino acid auxotrophs which we have used as recipients in the studies of transferable antibiotic resistance. The mutagenic system has been used in the past year to obtain metronidazole resisant mutants to investigate the mechanism of action of this drug. The usefulness of the bacteriophage typing system has been increased due to the isolation of additional phages obtained from Boston sewerage; studies over the past year indicate that over 90 percent of the strains of B. fragilis are typable.